Incidences of type 2 diabetes (T2D) have reached epidemic proportions, with ~8.3% of the US population diagnosed and 79 million more exhibiting pre-diabetes - a reminder of the urgent need for solutions. Halting pre-diabetes development and progression to T2D requires a multi-pronged approach, since the pathophysiology involves both peripheral insulin resistance and pancreatic ? cell dysfunction. Each of these processes is rate-limited by the abundance of exocytosis t-SNARE (Soluble NSF Attachment Protein Receptor) proteins. Relatedly, a recent human islet study suggests that increasing expression of exocytosis proteins, such as the t-SNARE protein Syntaxin 4 (Syn4), may lead to improved regenerative approaches to treat diabetes. Loss of Syn4 abundance and/or its activity is associated with diabetes in human and rodent islets and skeletal muscle, tissues which regulate insulin release and insulin sensitivity, respectively. Thus, the long-term goal is to understand how Syn4 can be manipulated for treatment and prevention of prediabetes and T2D. Discovery of ways to target Syn4 abundance/activation to control glycemic dysregulation and prediabetes offers a tantalizing opportunity for disease intervention. The objective of this application is to determine how Syn4 enrichment/activation functions to enhance ?-cell insulin secretion and skeletal muscle insulin action both in vivo and at the molecular level, and how Syn4 abundance in these tissues is regulated. Preliminary data show that Syn4 protein is limiting for human islet insulin secretion; islets enriched with Syn4 more effectively reduce hyperglycemia in diabetic mice. Moreover, T2D human islets are ~40% deficient in Syn4 protein, as are islet and muscle cells of diabetic mice. Notably, restoration of Syn4 to human T2D islets can fully rescue insulin secretion. Syn4-enriched mice also resist age-and high-fat-diet induced insulin resistance. The central hypothesis is that diabetogenic stimuli underlie Syn4 deficiency to impair key regulated exocytosis events in ? cells and skeletal muscle cells, and that Syn4 upregulation can boost these processes to rescue/resist stress associated with prediabetes and T2D. The rationale for the proposed research is that once it is known how Syn4 enrichment promotes and/or protects functional ? cell mass and peripheral insulin sensitivity, and how Syn4 abundance is regulated, that Syn4 can be manipulated to avert disease in the face of diabetogenic stimuli. Three Specific Aims are developed to test this: 1) Evaluate Syn4 upregulation in prevention/reversal of diabetogenic-induced ?-cell dysfunction, 2) Delineate how Syn4 enrichment/activation in skeletal muscle promotes insulin sensitivity, and 3) Determine the mechanistic basis for attenuated Syn4 expression in pre/diabetic tissues. Aims will be accomplished using innovative inducible ?-cell- and skeletal muscle-specific Syn4 transgenic mice challenged with diabetogenic stimuli, peptide activators of endogenous Syn4, and live-cell imaging biosensors paired with biochemical assays using human tissues. Results will positively impact efforts to ameliorate disease as the identified mechanisms are highly likely to provide new therapeutic targets.